The present invention is generally in the area of diagnostic imaging agents, and is particularly directed to microparticulate ultrasound imaging contrast agents having increased echogenicity and decreased attenuation as a function of the thickness of the polymer membrane.
When using ultrasound to obtain an image of the internal organs and structures of a human or animal, ultrasound waves, waves of sound energy at a frequency above that discernable by the human ear, are reflected as they pass through the body. Different types of body tissue reflect the ultrasound waves differently and the reflections that are produced by the ultrasound waves reflecting off different internal structures are detected and converted electronically into a visual display.
For some medical conditions, obtaining a useful image of the organ or structure of interest is especially difficult because the details of the structure are not adequately discernible from the surrounding tissue in an ultrasound image produced by the reflection of ultrasound waves absent a contrast-enhancing agent. Detection and observation of certain physiological and pathological conditions may be substantially improved by enhancing the contrast in an ultrasound image by injecting or infusing an agent into an organ or other structure of interest. In other cases, detection of the movement of the contrast-enhancing agent itself is particularly important. For example, a distinct blood flow pattern that is known to result from particular cardiovascular abnormalities may only be discernible by infusing a contrast-enhancing agent into the bloodstream and observing the dynamics of the blood flow.
Materials that are useful as ultrasound contrast agents operate by having an effect on ultrasound waves as they pass through the body and are reflected to create the image from which a medical diagnosis is made. Different types of substances affect ultrasound waves in different ways and to varying degrees. Moreover, certain of the effects caused by contrast-enhancing agents are more readily measured and observed than others. In selecting an ideal composition for a contrast-enhancing agent, one would prefer the substance that has the most dramatic effect on the ultrasound wave as it passes through the body. Also, the effect on the ultrasound wave should be easily measured. There are two effects which can be seen in an ultrasound image: backscatter and beam attenuation.
BACKSCATTER: When an ultrasound wave that is passing through the body encounters a structure, such as an organ or other body tissue, the structure reflects a portion of the ultrasound wave. Different structures within the body reflect ultrasound energy in different ways and in varying strengths. This reflected energy is detected and used to generate an image of the structures through which the ultrasound wave has passed. The term "backscatter" refers to the phenomenon in which ultrasound energy is scattered back towards the source by a substance with certain physical properties.
It has long been recognized that the contrast observed in an ultrasound image may be enhanced by the presence of substances known to cause a large amount of backscatter. When such a substance is administered to a distinct part of the body, the contrast between the ultrasound image of this part of the body and the surrounding tissues not containing the substance is enhanced. It is well understood that, due to their physical properties, different substances cause backscatter in varying degrees. Accordingly, the search for contrast-enhancing agents has focused on substances that are stable and non-toxic and that exhibit the maximum amount of backscatter.
The capability of a substance to cause backscatter of ultrasound energy depends on characteristics of the substance such as its ability to be compressed. When examining different substances, it is useful to compare one particular measure of the ability of a substance to cause backscatter known as the "scattering cross-section." The scattering cross-section of a particular substance is proportional to the radius of the scatterer, and also depends on the wavelength of the ultrasound energy and on other physical properties of the substance, J. Ophir and K. J. Parker, Contrast Agents in Diagnostic Ultrasound, Ultrasound in Medicine & Biology, vol. IS, n. 4, p. 319, 323 (1989).
In evaluating the utility of different substances as ultrasound contrast agents, i.e. gases, liquids, or solids, one can calculate which agents should have the higher scattering cross-section and, accordingly, which agents should provide the greatest contrast in an ultrasound image. It can be assumed that the compressibility of a solid particle is much less than that of the surrounding medium and that the density of the particle is greater. Using this assumption, the scattering cross section of a solid particle contrast-enhancing agent has been estimated as 1.75 (Ophir and Parker, supra, at 325). For a pure liquid scatterer, the adiabatic compressibility and density of the scatterer and the surrounding medium are likely to be approximately equal, which would yield the result that liquids would have a scattering cross-section of zero. However, liquids may exhibit some backscatter if large volumes of a liquid agent are present. For example, if a liquid agent passes from a very small vessel to a very large one such that the liquid occupies substantially all of the vessel, the liquid may exhibit measurable backscatter. Nevertheless, it is appreciated by those skilled in the art that pure liquids are relatively inefficient scatterers.
The scattering cross-section of a gas is substantially different and greater than a liquid or solid, in part, because a gas bubble can be compressed to a much greater degree than a liquid or solid. Moreover, free gas bubbles in a liquid exhibit oscillatory motion such that, at certain frequencies, gas bubbles will resonate at a frequency near that of the ultrasound waves commonly used in medical imaging. As a result, the scattering cross-section of a gas bubble can be over a thousand times larger than its physical size.
BEAM ATTENUATION: Another effect which can be observed from the presence of certain contrast-enhancing agents is the attenuation of the ultrasound wave. The intensity of the ultrasound wave decreases as the wave passes through the volume of tissue or blood containing the contrast agent. The decrease in wave intensity is the result of both ultrasound which is backscattered by the agent as well as dissipation of the wave as it interacts with the contrast agent. If the beam is too attenuated, the power returned to the transducer from regions distal to the contrast agent will be low leading to poor imaging depth. The use of beam attenuation differences in different tissue types has been attempted as an image enhancement method. Image contrast has been observed in conventional imaging due to localized attenuation differences between certain tissue types. K. J. Parker and R. C. Wagg, "Measurement of Ultrasonic Attenuation Within Regions selected from B-Scan Images," IEEE Trans. Biomed. Enar. BME 30(8), p. 431-37 (1983); K. J. Parker, R. C. Wagg, and R. M. Lerner, "Attenuation of Ultrasound Magnitude and Frequency Dependence for Tissue Characterization," Radiology, 153(3), p. 785-88 (1984). It has been hypothesized that measurements of the attenuation of a region of tissue taken before and after infusion of an agent may yield an enhanced image. However, techniques based on attenuation contrast as a means to measure the contrast enhancement of a liquid agent are not well-developed and, even if fully developed, may suffer from limitations as to the internal organs or structures with which this technique can be used. For example, it is unlikely that a loss of attenuation due to liquid contrast agents could be observed in the image of the cardiovascular system because of the high volume of liquid contrast agent that would need to be present in a given vessel before a substantial difference in attenuation could be measured.
In summary, diagnostic ultrasound is a powerful, non-invasive tool that can be used to obtain information on the internal organs of the body. The advent of grey scale and color Doppler imaging have greatly advanced the scope and resolution of the technique. Although techniques for carrying out diagnostic ultrasound examinations have improved significantly, as have those for making and using contrast agents, there is still a need to enhance the resolution of the imaging for cardiac perfusion and cardiac chambers, solid organs, renal perfusion, solid organ perfusion, and Doppler signals of blood velocity and flow direction during real-time imaging. The development of ultrasound contrast agents has focused on the use of biocompatible gases, either as free gas bubbles or as gases encapsulated in natural or synthetic shell materials.
A variety of natural and synthetic polymers has been used to encapsulate a gas, such as air, for use as imaging contrast agents. Schneider et al., Invest. Radiol., Vol. 27, pp. 134-139 (1992) describes 3 micron, air-filled polymeric particles. These particles were reported to be stable in plasma and under applied pressure. However, at 2.5 MHz, their echogenicity was low. Another type of encapsulated gas microbubble suspension has been obtained from sonicated albumin. Feinstein et al., J. Am. Coll. Cardiol., Vol. 11, pp. 59-65 (1988). Feinstein describes the preparation of microbubbles that are appropriately sized for transpulmonary passage with excellent stability in vitro. However, these microbubbles are short-lived in vivo, having a half life on the order of a few seconds (which is approximately equal to one circulation pass) because they quickly dissolve in under-saturated liquids, for example blood. Wible, J. H. et al., J. Am. Soc. Echocardiogr., Vol. 9, pp. 442-451 (1996). Gelatin-encapsulated air bubbles have been described by Carroll et al. (Carroll, B. A. et al., Invest. Radiol., Vol. 15, pp. 260-266 (1980), and Carroll, B. A. et al., Radiology, Vol. 143, pp. 747-750 (1982)), but due to their large sizes (12 and 80 .mu.m) they would not be likely to pass through pulmonary capillaries. Gelatin-encapsulated microbubbles have also been described in PCT/US80/00502 by Rasor Associates, Inc. These are formed by "coalescing" the gelatin.
Air microbubbles stabilized by microcrystals of galactose (SHU 454 and SHU 508) have also been reported by Fritzsch, T. et al., Invest. Radiol. Vol. 23 (Suppl 1), pp. 302-305 (1988); and Fritzsch, T. et al., Invest. Radiol., Vol. 25 (Suppl 1), 160-161 (1990). The microbubbles last up to 15 minutes in vitro but less than 20 seconds in vivo. Rovai, D. et al., J. Am. Coll. Cardiol., Vol. 10, pp. 125-134 (1987); and Smith, M. et al., J. Am. Coll. Cardiol., Vol. 13, pp. 1622-1628 (1989). Gas microbubbles encapsulated within a shell of a fluorine-containing material are described in WO 96/04018 by Molecular Biosystems, Inc.
European Patent Application No. 90901933.5 by Schering Aktiengesellschaft discloses the preparation and use of microencapsulated gas or volatile liquids for ultrasound imaging, where the microcapsules are formed of synthetic polymers or polysaccharides. European Patent Application No. 91810366.4 by Sintetica S. A. (0 458 745 A1) discloses air or gas microballoons bounded by an interfacially deposited polymer membrane that can be dispersed in an aqueous carrier for injection into a host animal or for oral, rectal, or urethral administration, for therapeutic or diagnostic purposes. WO 92/18164 by Delta Biotechnology Limited describes the preparation of microparticles by spray drying under very controlled conditions as to temperature, rate of spraying, particle size, and drying conditions, of an aqueous protein solution to form hollow spheres having gas entrapped therein, for use in imaging. WO 93/25242 describes the synthesis of microparticles for ultrasonic imaging consisting of a gas contained within a shell of polycyanoacrylate or polyester. WO 92/21382 discloses the fabrication of microparticle contrast agents which include a covalently bonded matrix containing a gas, wherein the matrix is a carbohydrate. U.S. Pat. Nos. 5,334,381, 5,123,414 and 5,352,435 to Unger describe liposomes for use as ultrasound contrast agents, which include gases, gas precursors, such as a pH activated or photo-activated gaseous precursor, as well as other liquid or solid contrast enhancing agents. WO 95/23615 by Nycomed discloses microcapsules for imaging which are formed by coacervation of a solution, for example, a protein solution, containing a perfluorocarbon. PCT/US94/08416 by Massachusetts Institute of Technology discloses microparticles formed of polyethylene glycol-poly(lactide-co-glycolide) block polymers having imaging agents encapsulated therein, including gases such as air and perfluorocarbons.
Although all ultrasound contrast agents investigated to date such as free gas bubbles or encapsulated gas bubbles are potent backscatterers, these agents also have a high degree of attenuation. High attenuation leads to low imaging depth and loss of tissue images distal to the contrast agent. In many cases, the imaging information can be lost completely beyond regions having significant concentrations of the contrast agent, e.g. the left ventricle. All ultrasound contrast agents currently under investigation share this problem to some extent.
To minimize the problem associated with the attenuation of contrast agents, investigators have resorted to several approaches. Most frequently the amount of contrast agent administered is decreased to allow more of the ultrasound beam to penetrate through the contrast agent. Although the attenuation is lower, the decrease in dose leads to less than optimal contrast for many clinical indications. Alternatively, ultrasound contrast agents can be administered as a continuous infusion. This essentially lowers the local concentration of agent and has the problem described previously for dose reduction. Continuous infusion has the additional disadvantages of requiring a larger total dose over time and is not easy to perform in a clinical setting. To compensate for lower doses, investigators have used harmonic imaging to enhance the signal to noise ratio. However, harmonic imaging is not standard at this point in time.
Importantly, these approaches do not address rectifying the fundamental problem with the acoustic properties of existing ultrasound contrast agents. Thus for an ultrasound contrast agent to have high echogenicity it is necessary to create a scatterer which leads to high total returned power at the receiving transducer from regions of interest at depths beyond the initial region containing contrast agent. The returned power will be governed by both the backscatter and the attenuation of the agent.
It is therefore an object of the present invention to provide microparticles with significantly enhanced echogenicity. It is another object of the invention to provide an ultrasound agent with high backscatter and low attenuation.